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Abstract
Recognition for the role of mtDNA in various diseases has grown over the past few decades since the first reports of diseases directly linked to mtDNA defects. However, treating such defects remains a challenge for several reasons not the least of which is a poor understanding of the mechanisms of mitochondrial DNA expression that is directly related to the current inability to routinely manipulate DNA inside the mitochondria in living mammalian cells. Developing such capability is dependent on the availability of delivery approaches as well as reporter constructs designed for exclusive mitochondrial expression. Hence, the aim of this project was to evaluate a novel 2.2kb linear DNA construct (Rgfp-FncrP) designed to express green fluorescent protein (GFP) as a potential reporter construct for mitochondrial gene delivery. The overall strategy involves delivering the construct into live mammalian cells using a mitochondria-specific STPP liposomal delivery system containing DOTAP, a DNA binding cationic lipid. Several highly sensitive detection protocols were then used to detect the expected increase in green fluorescence of the treated cells. The results indicated that the STPP liposomal formulation associated with cells, accumulated in the mitochondria, and could bind and deliver the construct to the mitochondria of live mammalian cells. In lipoplex transfected cells, a dose dependent increase in green fluorescence was observed which suggests functional expression of GFP. The results were further confirmed by flow cytometry and GFP-specific ELISA analysis. Though, the freeze-thaw, FT (2:1) lipoplex delivered more DNA to the mitochondria in live mammalian cells than regular mixing, RM (1:1) lipoplex, no significant difference between the two techniques were observed on comparing the GFP expression levels, suggesting that lipoplex preparation protocol has no influence on enhancing mitochondrial transfection efficiency.
